A gene carried on one of the multicopy plasmids commonly used for cloning and expressing foreign proteins in E. coli usually has a copy number of more than 20 copies/cell. Even low copy number plasmids (e.g., pACYC177 and pLG339) generally exist at 6-10 copies per cell. The expression of even minute amounts of some foreign proteins can kill host cells (see Meth. Enzymol. 185:63-65, ed. D. Goeddel, 1990). For this reason, it would be advantageous to reliably limit the copy number of genes encoding such toxic gene products, such as by integrating the gene into the bacterial chromosome at one or a small number of copies per cell. For example, such a system would allow one to make more representative cDNA expression libraries in bacterial hosts if the high-copy expression of one or more of the cDNAs in the library could kill the bacterial host or cause it to grow poorly.
Chromosomal integration of genes encoding heterologous polypeptides would also be advantageous as an alternative means for expression of foreign proteins in bacterial host cells. Multicopy vectors are often unstable and require the use of antibiotics in the growth medium for maintenance. Present methods of integrating foreign genes into the bacterial chromosome suffer from inefficiency, the inability to control the site of integration of the foreign gene, and/or the inability to control the copy number of the integrated gene. Most importantly, all efforts to date to create recombinant DNA constructs on the bacterial chromosome, wherein a bacterial promoter is fused to a heterologous gene, have involved the creation of viral or plasmid intermediates carrying the construct. Because such intermediates replicate at high copy number, they may be difficult or even impossible to recover in cases where the foreign gene product is toxic to the bacterial cell.
Previous methods for achieving the integration of foreign genes into the chromosome of a bacterial host include the use of phage .lambda. vectors. The phage DNA in circular form is inserted linearly into the bacterial chromosome by a single site specific recombination between a phage attachment site (att P), 240 bases long, and a bacterial attachment site (att B), only 25 bases long. The two sites have 15 bases in common. This site-specific recombination event is catalyzed by a special integrase, specified by the phage gene INT (Virology, 2nd ed., R. Dulbecco and H. Ginsberg, eds., Philadelphia: Lippincott, pp. 56-57 (1985)).
Phage vectors which are INT.sup.- can be integrated into the chromosome in a normal fashion as long as integrase is supplied in trans, e.g., by an INT.sup.+ helper phage (see, e.g., Borck et al. (1976) Molec. Gen. Genet. 146:199-207).
Phage vectors which are both att.sup.- and INT.sup.- can likewise be integrated into the bacterial chromosome as double lysogens by using att.sup.+INT.sup.+ helper phage. Double lysogens formed by linkage of the prophages at the bacterial attachment site are integrated into the chromosome by general bacterial recombination between homologous sequences on the defective phage and on the helper phage (see, e.g., Struhl et al. (1976) Proc. Natl. Acad. Sci. USA 73:1471-1475). Similarly, it is also possible to integrate non-replicating colE1 replicons into the genome of polA strains of E. coli by means of recombination between the host chromosome and homologous sequences carried by the plasmid vector (Greener and Hill (1980) J. Bacteriol. 144:312-321).